Leptospirosis is an important, global human and veterinary health problem. It is a widespread zoonotic disease caused by pathogenic strains of Leptospira species which are capable of infecting most mammalian species. Infection occurs either through direct contact with an infected animal or indirect contact with contaminated soil or water. In livestock, the disease causes economic losses due to abortion, stillbirth, infertility, decreased milk production, and death.
Efforts to control leptospirosis have been hampered because virulent leptospires have the capacity for both long-term survival in the environment as well as persistent infection and shedding by wildlife and livestock. Currently available leptospiral vaccines produce short-term immunity and do not provide cross-protection against many of the 170 serovars of pathogenic Leptospira species {Thiermann, et al., J. Am. Vet. Med. Assoc., 184:722 (1984)}. These vaccines consist of inactivated whole organisms or outer envelope preparations which produce seroreactivity as determined by microscopic agglutination of intact organisms. The nature of the protective immunogens in these vaccine preparations has not been conclusively elucidated, although several lines of evidence suggest that lipopolysaccharide-like substance ("LLS") may confer a degree of protection. Commercially available vaccines, which consist of heat or formalin-killed leptospires, produce incomplete or only short-term immunity, requiring their administration annually or semiannually. In the case of L. interrogans serovar hardjo, the common bovine pathogen in North America, vaccines prepared in this way are ineffective {Bolin, C. A., et al., Am. J. Vet. Res., 50:161-165 (1989) and Bolin, C. A., et al., Am. J. Vet. Res., 50:2004-2008 (1989)}. Thus there is an important need for development of an improved leptospiral vaccine.
The pathogenesis of leptospirosis is similar to that of other spirochetal diseases, including syphilis (caused by Treponema pallidum) and Lyme borreliosis (caused by Borrelia burgdorferi). Both syphilis and Lyme borreliosis are characterized by widespread dissemination early in the course of disease, including invasion of the central nervous system. Leptospira species share this ability with other pathogenic spirochetes such that meningitis is a common manifestation of leptospirosis. Another feature of spirochetal infections is the ability to persist chronically in the host, as manifested in cases of tertiary syphilis and chronic Lyme arthritis.
Identification of outer membrane ("OM") components is essential in the development of protective immunogens for spirochetal diseases. There are at least two classes of leptospiral outer membrane proteins ("OMPs"). One class of leptospiral OMPs are the transmembrane proteins, such as the porin OmpL1 and the TonB-dependent OmpL2, that are produced in small amounts by pathogenic Leptospira species {Haake, D. A., et al., J. Bacteriol., 175:4225-4234 (1993); Haake, D. A., et al.}. Transmembrane OMPs are distinguished from other membrane proteins structurally by the fact that they contain beta-sheet membrane-spanning regions. They are also functionally unique in that they create channels for transport of nutrients across the outer membrane.
A second class of leptospiral OMPs are the lipoproteins, which are produced by Leptospira species in generous amounts. Lipoproteins are anchored to membranes by fatty acids attached to their amino-terminal cysteine. Both the outer membrane and cytoplasmic membrane contain lipoproteins, and the signal(s) by which lipoproteins are translocated to the outer membrane are not known. Therefore, in order to define a lipoprotein as an OMP, it must be shown to be a component of isolated leptospiral OM. Several Triton X-114 detergent phase lipoproteins have been identified, including LipL36 (also referred to as "LipL1") and LipL41 (also referred to as "LipL2"). LipL1 and LipL2 are described in Shang, E. S., et al., "Molecular Cloning and Sequence Analysis of the Genes Encoding Two Leptospiral Lipoproteins, LipL1 and LipL2", Abstract No. D-2, in Abstracts of the Annual Meeting of the American Society for Microbiology, May 21-25, 1995, p. 249 (American Society for Microbiology, Washington, DC, 1995), this reference is herein incorporated by reference in its entirety. The Triton-extractable 32-kDa major outer membrane protein is probably also a lipoprotein {Zuerner, et al., Microbial Pathogenesis, 10:311-322 (1991)}. However, lacking a carefully defined technique for isolating the leptospiral OM, it was not possible to determine to what extent these lipoproteins are found in the OM, nor to identify additional components of the leptospiral OM.
Development of techniques for isolation of the OM from Leptospira species and other spirochetes has been difficult because of their unique architecture {Holt, S. C., Microbiol. Rev., 42:114-160 (1978)}. Like enteric gram-negative bacteria, spirochetes have both an outer membrane and a & cytoplasmic membrane, separated by a periplasmic space. However, spirochetal architecture differs significantly from that of gram-negative bacteria in that the peptidoglycan cell wall of spirochetes is associated with the cytoplasmic membrane rather than the outer membrane. For this reason, the spirochetal outer membrane is extremely labile and difficult to isolate from components of the underlying cell wall and cytoplasmic membrane which together constitute the protoplasmic cylinder ("PC").
A number of approaches have been used in the isolation of the leptospiral outer membrane {Auran, et al., Infect. Immun., 5:968-975 (1972); Nunes-Edwards, et al., Infect. Immun., 48:492-497(1985); Nicholson, et al., Veterinary Microbiology, 36:123-138 (1993)}. These studies did not take into account spirochetal outer membrane fragility and the lack of OM selectivity of ionic or nonionic detergents {Penn, et al., J. Gen. Microbiol., 131:2349 (1985); Stamm, et al., Infect. Immun., 55:2255 (1987); and Cunningham, et al., J. Bacteriol., 170:5789 (1988)}. Notably lacking from these reports are controls to assess contamination of the OM fraction with PC components. Recently, spirochetal OM isolation has been advanced by developments in three areas. Firstly, specific OM markers such as porins (e.g. OmpL1) have been identified, allowing assessment of efficiency of OM release. Secondly, techniques have been developed for assessing the degree of contamination with PC components. Thirdly, new techniques involving hypotonic citrate and hypertonic sucrose have been found to be of use in the isolation of the OM of Treponema species {Blanco, et al., J. Bacteriol., 176:6088-6099 (1994); Radolf, et al., Infect. Immun., 63:2154-2163 (1995)} and B. burgdorferi {Skare, et al., J. Clin. Invest., 96:2380-2392 (1995); Radolf, et al., Infect. Immun., 63:4244-4252 (1995)}. Without modification, these techniques did not facilitate isolation of the leptospiral OM.